500g 1kg 10kg 500g 1kg 10kg -...

Our research convincingly argues that:1: the best indicator of age is the abundance of short telomeres, 2: even weak Telomerase inducers such as TA-65 will increase the abundance of short telomeres, 3: that family 5 compounds are safe for use on skin.4: Telomerase has an anti-aging effect on skin.5: Family 5 compounds are much stronger than TA-65.

Several years ago Sierra Sciences created and patented what is the world’s most effective telomerase inducing compounds. This compound is perfect for immediatesale to the cosmetics industry. There are 6 of these molecules, all very similar in structure and function. We have named them Family 5. We believe Family 5 will beof great value to a multi-national skincare company which can claim, with proven scientific evidence, to exclusively have the only telomerase inducing ingredient in their products. Our Family 5 molecules have been tested on human cells. In fact, allof the data presented is the result of testing on human cells both to determine effectiveness at inducing telomerase and to prove safety and suitability for use in topical application. This is why we are certain that these compounds will be a valuable asset to a skincare company.

Sierra Sciences have been basically sitting on a goldmine without realizing it. It was not until the recent buy out of Sierra Sciences By Andrews Inc and a more forward thinking way of evaluating things that the right questions were asked and the conclusion reached that not only are these molecules commercially viable they are VERY profitable due to the concentration at which they are most effective with just 1g of our chemicals delivering 100litres of full potency topical applications. These molecules are seriously for sale today! With the option for exclusive rights, too!

Sierra Sciences is looking to sell these molecules to a large, successful, science-based skincare company. Our goal is to sign an exclusive agreement with a major skin care company with sales of over $200,000,000 per annum and to have a similarstructure as we do with Isogenix whereby we receive a percentage of the retail value of their products as payment for and exclusive rights to our molecules.

Non GMP

GMP grade

500g 1kg 10kg 500g 1kg 10kg

CO314813

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Lead time (weeks)

4-6 6-8 8-10 8-10 8-10 12-15

CO314814Lead time (weeks)

5-7 8-10 10-12 10-12 10-12 15-17


4-6 6-8 8-10 8-10 8-10 12-15


5-7 8-10 10-12 10-12 10-12 15-17


5-7 8-10 10-12 10-12 10-12 15-17


5-7 8-10 10-12 10-12 10-12 15-17

UNDERSTANDING THE DATA

Our cells undertake distinct steps to express a gene (that is, use our genetic blueprint to create a protein, like telomerase). The first step is “transcription”, where messenger RNA is made from the DNA blueprint. The final step is “translation,” where the cells make a protein using the instructions in the mRNA. If we can confirm that we have both the first and last step working, it is a reasonable assumption that the compound is doing exactly what we want it to do.

Our hTERT mRNA assay is a way for us to observe transcription initiation. We isolate the messenger RNA and make millions of copies of it using PCR, a technique that amplifies DNAor RNA, so that it can be measured. We run PCR until the telomerase mRNA reaches a specific, measurable threshold. Then, by counting the number of PCR amplifications we had

TELOMERASE EXPRESSION, SENESCENCE, AND SKIN FUNCTION 3

to do, we can mathematically extrapolate how much there was to begin with.

The first chart shows that the telomerase gene was transcribed and processed correctly. When we ran PCR, we indeed saw a strong signal of telomerase mRNA. On the y-axis, we’remeasuring mRNA based on a unit called “Ct.” That is the number of times we had to amplify the mRNA before it reached our specific threshold. The more mRNA that was there to begin with, the fewer Ct it takes to reach that threshold. So, when the Ct is a smaller number, that means there was more mRNA present to begin with.

In a nutshell, the y-Axis shows how much mRNA our compound induced. The taller the bar, the stronger the compound when it comes to inducing transcription initiation.

The yellow bars show how toxic the compound was by showing what percent of living cells were counted after exposure to the compound. The lower the bar, the more toxic the compound is. As you can see, C0314818 is not very toxic at all. On the right of the graph is our positive control, C0057684, the first telomerase inducer we discovered, and the one we’ve most thoroughly studied. The chart shows that C0314818 is about three times stronger than C0057684.

The second chart is TRAP data. TRAP measures protein activity and shows that translation, the final step in gene expression, successfully occurred. In TRAP, we’re directly measuring how much telomerase protein is present. So, we know that our compound both began and finished gene expression. It presumably went through all the steps.

TRAP works using a technique called gel electrophoresis. Gel electrophoresis can sort DNA molecules based on size, and display them as distinct bands on a gel. Telomerase is a unique enzyme, in that it lengthens telomeres 6 bases at a time, so when you sort the molecules into bands on the gel, you see a repetitive sequence of 6 basis, forming a “ladder” pattern. If you see a ladder, you know you have telomerase. And the darker the ladder is, the more telomerase is present.

The TRAP data on C0314818 shows that we see activity at concentrations from 3.70 micromolar up to 33.33 micromolar. Again, TRAP confirms that C0314818 is about three times stronger than C0057684 at a concentration of 33.33 micromolar. We measure the intensity of the bands with a tool called a spectrophotometer.

Our control for TRAP is a cell line called HeLa, which produces what we believe to be the minimum amount of telomerase necessary to make a human cell immortal. The bands

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created by C0314818 are 1.6 times as strong as bands created by measuring a mixture that is composed 10% of HeLa cells. Therefore, we conclude that C031818 causes a cell to produce about 16% of the telomerase that it would take to make that cell immortal.

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We commissioned Abich laboratories S.r.l. - based in Lago Maggiore. Northern Italy. 150 grams of each sample to Dr Elena Bocchietto (Abich Laboratories) for thefollowing evaluations:

SYNOPSISTESTING of NEW COMPOUNDS (SMALL MOLECULES)There are proper procedures for determining proof of concept - Lead compounds go through a series of tests to provide an early assessment of the safety of the lead compound. Scientists test Absorption, Distribution, Metabolism, Excretion and Toxicological (ADME/Tox) properties, or “pharmacokinetics,” of each lead.Successful compounds (drugs) must be:• Absorbed into the bloodstream,• Distributed to the proper site of action in the body,• Metabolized efficiently and effectively,• Successfully excreted from the body and• Demonstrated to be not toxic.

In this instance, as a starting point, very rudimentary safety profile and risk assessment testing was carried out SAMPLE PREPARTION The compounds were used at 0.25% w/w. This value of 0.25% was selected randomly but on the lower side first and foremost because of its unknown nature. Stereotypicallytopical treatments have active components loaded at anywhere from 0.01% to 2% to achieve efficacious functionality.

1. Synthetic compounds as supplied by Federico Gaeta - L031272-02 & L0314813-02

2. L031272-02: Appears to be only partially water-soluble. There are various options for achieving improved product stability and skin delivery. In this situation we selected liposomal delivery. The active was encapsulated into a multilamellar vesicle liposome then added to the emulsion as per normal production parameters.

- The Liposomal ingredients consisted of Phosphatidylcholine, Hydrogenated Lecithin, Glycerin and Alcohol.

- Encapsulation was achieved by microfluidisation – Although this was notmeasured or sized a milky solution without separation was realized during the process. This normally is evident of liposome formation.


- Loading at 10% min (~ 12-15%)

3. L0314813-02: This compound appears to be water dispersible or at least predominately soluble – This was pre-dispersed in water at 30°C then immediately added to the emulsion at the final stage under homogenization.

4. Both Actives were then introduced to their respective emulsion systems at temperatures below 30°C

5. The Emulsion system used in both test cases was a Lipid Glucoside system. Which could be deemed as an advanced emulsion system – it provides a liquid crystal structured emulsion (Anisotropic fluids). From experience we know this system provides 1-5 micron size droplets with excellent stability and superior dermal delivery.

6. The Emulsion Ingredient List: Water – (emulsifiers) Cetearyl Glucoside - Arachidyl Glucoside – Behenyl, Arachidyl, Cetearyl Alcohols - (Emollients’) C12-15 Alkyl benzoate – Diethylhexyl succinate – Jojoba Oil – Almond Oil – (Humectant) Glycerin – (Preservatives) Caprylhydroxamic Acid - Glyceryl Caprylate

7. 200 gram samples prepared with the active compound added to ensure 0.25% w/w of respective compounds - L0314813-02 When added a drop in viscosity was evident but did not require adjustments – Final pH 5.6 - L031272-02 When added no dramatic change was noted – Final pH 5.7

- T023777-02 is a PLACEBO/BLANK – Cream Base only - minus any active compounds

8. Stability – At this stage a small 20-gram sample of each was retained for real time stability testing or observation. Currently there are no test protocols developed to determine the compound behavior or quantitative measurement in emulsion systems. Obviously it is too early to comment on stability. In the cosmeceutical arena we only measure emulsion stability by microbiological and chemical parameters. Normally physical and rheological changes are evidence of emulsion instability – Be it preservative failure or

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internal physical and chemical structural failure. Once commercial products are developed a dedicated stability program would be required – Guidelines would be based on PAO (Period after opening) This would require 30 months shelf life

BACK GROUND INFORMATION As discussed previously – Initially the products are to be promoted as cosmeceuticals, and not medicine/medical devices, which basically mean far less stringent testing and regulatory constraints. Where applicable or used, a normative reference has been provided.

However: As per the Council Directive 76/768 and the Regulation (EC) no 1223/2009, any cosmetic product marketed or sold within the Community must notcause damage to human health when applied under normal or reasonably foreseeable conditions of use. To this purpose specific studies are performed to protect both the manufacturer and the end consumer. After performing toxicological evaluations and specific in-vitro preliminary tests, a competent specialty laboratory can assess the tolerability of a cosmetic product with tests on human subjects.These tests are normally conducted under strict supervision of an investigator on panelists who are enrolled according to the following inclusion criteria: (a) good state of health, (b) no skin disease, (c) no pharmacological treatment in progress, (d) promise not to change one’s daily routine (e) negative anamnesis for atopy. All the partnership facilities that we work with follow the Ethical Principles for Medical Research Involving Human Subjects written in the “World Medical Association Declaration of Helsinki 2008”

We commissioned Abich laboratories S.r.l. - based in Lago Maggiore. Northern Italy. 150 grams of each sample to Dr Elena Bocchietto (Abich Laboratories) for the following evaluations: Cytotoxicity (in-vitro) UNI EN ISO 10993-5: 2009

Dermal Irritancy & Sensitisation,

Patch Testing,


Please note: ALL testing facilities require disclosure of active compounds being tested – I did not disclose details or any information as to the source of these compounds – For the purposes of this testing, they were registered as “Peptides”

Where applicable or used, a normative reference has been provided.CYTOTOXICITY - UNI/ENISO10993-5:2009Aim of the test is to evaluate the cytotoxicity of finished products or raw materials intended to be used on the skin or on the mucosae following the UNI EN ISO 10993-5 standard concerning the biological evaluation of medical devices. The MTT assay determination assay is simple, accurate and yields reproducible results. Mossman originally developed this method.

The cytotoxicity assay performed in this study was designed to evaluate the cytotoxic potential of the tested product on the fibroblasts/ keratinocytes. .This method is customized for improved result interpretation based on historical results obtained in this particular laboratory with an improved non-standard keratinocyte cell line.

The key component is (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)or MTT. This product is of yellowish colour in solution. Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, leading to the formation of purple crystals that are insoluble in aqueous solutions. The crystals are re-dissolved in isopropanol and the resulting purple solution is measured spectrophotometrically.An increase or decrease in cell number results in a concomitant change in the amount of formazan formed, indicating the degree of cytotoxicity caused by the test material.

Cells have been seeded in 96 wells plates (10000cells/well) and allowed to grow for 24 h at 37°C and 5% CO2.The second day fresh medium is added, supplemented with 6 scalar dilutions of the tested product ranging from 5.00 mg/ml μl/ml and 0.16 mg/ml μl/ml. The sample has been dissolved directly in the medium culture. The test is carried out in three replicas for each test dilution. At the end of the incubation period, the cells are tested for their viability with the cytotoxicity (MTT) assay. Cells treated with a known irritating surfactant (Sodium Lauryl Sulfate – SLS) in concentration ranging from 0.5mg/ml to 0.03mg/ml are used as positive control. Untreated cells are used

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as blank control. The MTT assay allows to evaluate the toxic impact of thetested compound on the cells viability.

After 24h exposure of the cells to the test material, the culture medium is removed and the cells incubated for 2 h in 100 μl/well of 1mg/ml MTT solution at 37°C. The solution is then removed and replaced with 200 μl/well of isopropanol, with further30’ incubation at room temperature under medium speed shaking.The absorbance at 570 nm is measured with a microplate reader (Tecan modello Sunrise remote), deducting background at 650 nm. The absorbance values are corrected by subtracting the reading of the blanks, with the diluent only.

The results are expressed in terms of viability:% Of cell viability = [OD(570 nm - 650 nm) test product / OD(570 nm - 650 nm) negative control] x 100In that case it is possible to calculate the IC50 value (Inhibiting Concentration 50), which indicates the concentration of the test compound, which inhibits the cell viability of 50%.For finished products, IC50 values higher than 1mg/ml (or 1μl/ml) may be considered as irritating, values lowest than 3mg/ml (or 3μl/ml) show a good biocompatibility. For detergents and rinsing off products, the eventual dilution factor should be taken in account. For raw materials, the use concentration should be taken in account. (Translated from Italian)

RESULTS

Compound IC50/ IC50 values Explanation

L031272-02 > 5mg/ml Very good biocompatibility-not cytotoxic

L0314813-02 > 5mg/ml Very good biocompatibility-not cytotoxic

T023777-02 > 5mg/ml Very good biocompatibility – not considered cytotoxic

SKIN IRRITATION OECD/OCDE: Guidelines for the testing of chemicals In-vitro Irritation on reconstituted human epidermis test methodSkin irritation and skin sensitization are different types of reactions… the first is caused by irritating agents (such as SLS) and it may happen after the first contact with the


irritating substance. The sensitization is a more complex reaction in which immune system is involved (repeated exposure to the allergens are needed to evoke the activation of the immune system).1. The test chemical is applied topically to a three-dimensional RhE model, comprised of non-transformed human-derived epidermal keratinocytes, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous tothose found in vivo.2. Chemical-induced skin irritation, manifested by erythema and oedema, is the result of a cascade of events beginning with penetration of the stratum corneum and damage to the underlying layers of keratinocytes. The dying keratinocytes release mediators that begin the inflammatory cascade that acts on the cells in the dermis, particularly the stromal and endothelial cells. It is the dilation and increased permeability of the endothelial cells that produce the observed erythema and oedema (24). The RhE-based test methods measure the initiating events in the cascade.3. Cell viability in RhE models is measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1], into a blue formazan salt that is quantitatively measured after extraction from tissues (25). Irritant chemicals are identified by their ability to decrease cell viability below defined threshold levels (i.e. ≤ 50% for UN GHS Category 2). Depending on the regulatory framework and applicability of the Test Guideline, chemicals that produce cell viabilities above the defined threshold level, may be considered non-irritants (i.e. > 50%, No Category).RESULTS

Compound Results

L031272-02 Potential threshold > 50%

L0314813-02 Potential threshold > 50%

T023777-02 > 50%

SKIN SENSITISATION Monocyte-derived human Immature Dendritic Cells as IN VITRO Models for predicting potential sensitizing of chemicals.

It is now well established that dendritic cells (DC) play pivotal roles in the initiation and orchestration of adaptive immune responses, including cutaneous immune responses to chemical allergens that drive the acquisition of skin sensitization. It is not unexpected; therefore, that a large number, and wide variety, of proposed approaches for the identification of skin sensitizing chemicals in vitro are based upon

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the use of cultured DC or DC-like cells. The use of DC in this context is legitimate. However, with our rapidly increasing understanding of the diversity of cutaneous DCwith respect to both phenotype and function, it is timely now to review briefly the potential limitations and interpretive difficulties that are associated with the use of DC-based assays. Among the important considerations are the fact that chemical-induced changes in the characteristics and function of cultured DC will not necessarily reflect accurately the events that that support the development of skin sensitization in vivo. In addition, most DC-based assays are predicated on a view thatcutaneous DC have as their primary function the initiation of adaptive immune responses. However, it is now appreciated that cutaneous DC, and in particular epidermal Langerhans cells (LC), may also play important immunoregulatory roles that serve to limit and contain skin immune responses. Notwithstanding these considerations there is reason to believe that at least some in vitro DC-based assays are of value, and indeed some are currently the subject of a formal validation process. However, it is appropriate that such assays are configured and interpreted carefully, and with an appreciation of the complexity of DC biology.

RESULTS

L031272-02 As tested deemed not Sensitizing

L0314813-02 As tested deemed not sensitizing

T023777-02 As tested deemed not sensitizing

Read More: http://informahealthcare.com/doi/abs/10.3109/15569527.2012.692135?journalCode=cot

Human skin equivalent / reconstituted epidermis culturesIn vitro three-dimensional cultures of living human skin generally fall into two categories: epidermal or skin equivalents. Epidermal equivalents consist of keratinocytes which, when cultured on a filter or matrix at the air-liquid interface, develop into a fully differentiated epidermis with a stratum corneum. Kubilus et al. (1996) examined cell viability, histological changes and the release of IL-1 and prostaglandin E2 (PGE2) by the EpiDerm model following treatment with irritants and allergens. They found that the dose response curves for the release of IL-1 and PGE2 following treatment with contact irritants reflected the cytotoxicity of the dose(i.e. the higher the cytokine release, the greater the cytotoxicity).However, following treatment with contact allergens, the amount of cytokine released did not correspond directly to the degree of cytotoxicity as more IL-1 and PGE2 were produced at non-cytotoxic doses of the chemical and varied with the

http://informahealthcare.com/doi/abs/10.3109/15569527.2012.692135?journalCode=cot

http://informahealthcare.com/doi/abs/10.3109/15569527.2012.692135?journalCode=cot


allergen. The SkinEthic model was used by Coquette et al. (1999) to measure IL-1 and IL-8 mRNA and protein levels as well as cytotoxicity following irritant and allergen treatment. More recently, Coquette et al. (2003) have suggested that determination of IL-8, with IL-1, and MTT conversion shows potential to discriminate and classify irritants and allergens in a single assay. Like Kubilus et al., they report that IL-1 release increases with cytotoxicity after treatment with the irritants Triton X100 and BC, but had no effect on IL-8 levels. In contrast, the allergen DNCB did not induce IL-1 but instead, elicited elevated IL-8 levels. However, when mRNA expression was measured, BC, Triton X100 and DNCB all induced an up-regulation in message for IL-8 while only BC up-regulated IL-1 mRNA.

The results of a number of studies conducted to assess the ability of irritants and contact allergens to affect cytokine mRNA levels in skin equivalents have been reviewed by Gerberick and Sikorski (1998). Data from those studies indicated that while cytokine message could be modulated by chemical treatment, each chemical appeared to produce changes in cytokine mRNA expression that was unique to the chemical and was also concentration and or time-dependent. No single cytokine or profile of cytokines was identified which were predictive for sensitization potential. However, Corsini et al. (1999) recently reported that interleukin-12 (IL-12) is selectively induced in the Episkin model by treatment with chemical allergens. Following a 3 hour exposure to test chemicals, mRNA levels for both IL-12 p40 and IL-12 p35 were found to be up-regulated by contact allergens (oxazolone, eugenol and DNCB) but not the irritants (SLS and BC).

1. References - See body of the text2. Developer of the method - Not applicable3. Known users - None4. Status of validation and/or standardisation – No validation of test methods to date.5. What efforts are needed to complete validation of the method not applicable

PATCH TESTING SKIN IRRITATION TEST - Patch Test (Irritation and delayed-type hypersensitivity)

The evaluation of a potential irritant effect of a cosmetic product according to the amended Draize method. The scope of this test is to evaluate the tolerability of a cosmetic product by determining and classifying its potential irritant effect. This test may support the ‘dermatologically tested’ claim.

20 healthy volunteers – Female and Male between the age of 18 and 65 Over a 72 hour period

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RESULTS

Compound MII (Mean Irritation Index) Classification

L031272-02 ≤ 0.4 (> 80%) Non Irritant

L0314813-02 ≤ 0.4 (> 70%) ≤ 0.5 – 1.9 Non irritant

T023777-02 ≤ 0.4 (>70%) ≤ 0.5 – 1.9 Non Irritant

Human Repeated Insult Patch Tests (HRIPT)Short description, scientific relevance and purposeThe HRIPT is performed in at least four different forms. The concentration of material chosen for induction and challenge in the HRIPT is determined by considering the following factors: previous human experience, previous sensitisation tests in guinea pigs and irritation studies in humans. It is common practice to test multiple substances simultaneously, because it saves time and cost, but the scientific basis for multiple simultaneous inductions is not substantiated.a) Draize test: ten consecutive induction patches are applied to new skin sites on the arms or back for 24 hours every other day 3 times a week. Each induction site is evaluated for erythema and edema after removal of the patch. Two weeks after the last induction, a challenge patch is applied for 24 hours and subsequently read. The response after challenge is compared to the responses reported after the early induction patches.b) Shelanski-Shelanski test: it is comparable to the original Draize HRIPT but employs 15 consecutive induction patches to the same site and if erythema and/or edema develops during induction the following patch should be moved to an adjacent untreated area. 2-3 weeks after the last induction a challenge patch is applied for 48 hours and scored. The induction patch responses are also noted and interpreted as evidence of cumulative irritation.c) Voss-Griffith test: it is also like the original Draize HRIPT with nine 24-hour patch tests conducted over a 3 weeks period and challenge is performed 2 weeks later with duplicate patches applied to the induction skin site and to the opposite arm.This assay allowed testing of four materials simultaneously. Repeated challenge is recommended in case of dubious reactions.d) Modified Draize test: it differs from the original Draize test by subjecting the volunteers to a continuous induction period with patch exchange 3 times a week untila total of 10 patches have been applied. The patches are reapplied to the same site, and only if moderate inflammation has developed, the next patch is moved to an


adjacent skin site. Challenge is performed on naive skin two weeks later with a 72-hour patch test with a non-irritating concentration of the substance.Nowadays, the HRIPT is usually only performed to confirm the safe use of potentiallysensitizing substance in consumer products, such as cosmetics or household products. Other substances are not normally tested in the HRIPT. The test concentration is normally at the upper end of the suggested use concentration range and is below concentrations giving positive results in animal tests.

Referencesa) Draize et al., 1944, Draize, 1959b) Shelanski and Shelanski, 1951; Shelanski, 1953c) Voss, 1958, Griffith and Buehler, 1976d) Marzulli and Maibach, 1973

FOR CONSIDERATION and DISCUSSION The tests show that the 2 compounds suggest that in the first instant they are

innocuous and well tolerated. We are not aware of any stability of the Synthetic compounds once

incorporated into an emulsion system

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FIG. 3. Mean telomere length and percent of nuclei with short telomeres at baseline and post TAS protocol. (A) Mean telomere length values standard error (SE) of the indicated individuals at base line (black bars)

and post PattonProtocol-1 (grey bars) as determined by high-throughput quantitative fluorescent in situ hybridization (HT qFISH). The total number of nuclei analyzed is indicated (n) on top of each bar. Statistical significance was assessed by the Student t-test. (B) Percentage of nuclei with short (<4 kb) telomeres at base line (black bars) and post-TAS protocol (grey bars) as determined by high throughput (HT) qFISH. Numbers above bars represents the number of nuclei with short telomeres (<4 kb) out of the total number of nuclei analyzed. The chi-squared test was used to evaluate the statistical significance for each individual tested.

Discussion and Conclusions


The inability to maintain telomeres with age and chronic stress has been linked to declining health and the increased risk of disease and death from many causes, including cancer.16,25,49–52 In this study, we report that a 1-year health maintenance program consisting of a dietary supplement pack combined with a natural product–derived telomerase activator results in a decreased percentage of short leukocyte telomeres and remodeling of the relative proportions of the circulating leukocytes of CMVþ subjects toward the more ‘‘youthful’’ profile of CMV subjects.One of the strengths of our study is the low CMVpositivity rate (54%) in a relatively older population, which allows us to separate the effects of age and CMV status on immunosenescence. It also serves to mitigate one of our study’s weaknesses—the lack of a control group—as the subjects were initially unaware of their CMV status and their subsequent knowledge is unlikely to have caused the segregation of manyof the effects of the protocol by CMV status.Our description of the decrease in CD8þCD28 T cells as a positive remodeling of the immune system is supported by the increased morbidity and mortality associated with what is known as the immune risk profile (IRP). This profile has been defined as a CD4/CD8 less than 1 in association with CMV seropositivity by longitudinal studies of individuals in their eighties and nineties in the OCTO/NONA Swedish cohort.53 As in our cohort, the major driving force for the decreased CD4/CD8 in CMVþ subjects in this population is likely the accumulation of virus-specific CD8þCD28 T cells. These studies have reported 6-year follow-up data, and no individuals who have survived to 100 years old exhibit the IRP, even if they are CMVþ. The authors conclude that successful immune aging entails being able to control CMV infection without accumulating senescent cytotoxic T cells. Thus, we conclude that the 20% reduction in CD8þCD28 T cells is a salutary effect, even though we have yet to see increases in the number of CD8þCD28þ T cells. Telomere shortening associated with replicative senescence is the probable cause of loss of CD28 expression and apoptosis resistance of CD8þ T cells.54 The decrease in the percentage of short telomeres we found makes upregulation of telomerase by TA-65 the most likely mechanism for this salutary effect.Age-related changes in the innate immune system have not been as well characterized as those of the adaptive immune system, although the importance of changes in the former is increasingly being recognized.55 It is generally agreed that the per-cell activity of neutrophils as measured by oxidative burst, phagocytosis, and chemotaxis decreases with age.56 There is less agreement on the effect of aging on neutrophil number which has been variously reported to be preserved,57

decreased,58 or increased53 with age. CMV status is not reported in the first two studies, but in the last study from the above-mentioned NONA cohort, the increase in neutrophil number is based on a comparison between 18 middle-aged (55-year-old) subjects with a 55% CMVþ prevalence rate and 120 very old subjects (92–100-

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year-old) with a 87% CMVþprevalence rate. Most of the cross-sectional increase of 960 neutrophils occurs within the 92 to 100 year olds, with only a 52-cell increase between 55 and 92. There is also a significant longitudinal increase over a 6-year interval in the very old group. This suggests that there is selection for those very oldsubjects able to increase their circulating neutrophils in the face of deteriorating tissues, increased inflammation, and increased exposure to infectious agents. Our novel finding that by age 62 the neutrophil number is 20% higher and continues to increase with age only in CMV subjects can be interpreted as a compensatory increase in the face of declining per cell activity and barrier function, as well as increased antigenic load. The absence of a cross-sectional increase with age in neutrophil number in CMVþ subjects suggests that this compensation is blocked in the CMVþ subjects perhaps due to inhibitory cytokine production by the senescent Tcells. The effect of the protocol to increase neutrophil count in CMVþ subjects can be interpreted as a salutary removal of this block in part through reduction in the number of senescent T cells.There is a broad consensus that NK cell number increases with age to compensate for decreased per-cell activity, which results from impaired signal transduction,59

but other mechanisms such as decreased barrier function and increased antigenic/pathogenic load may also contribute to increased NK cells with age.60 The decrease in NK cell number induced by the protocol is an ‘‘age reversal,’’ but because we did not measure NK activity we cannot say whether it is from improved barrier function or improved signal transduction. Unlike other cells of the innate immune system, NK cells proliferate after activation and experience further telomere shortening once they are released from the bone marrow.61 The decrease in the percentage of short telomeres we found could result in improved signal transduction as a mechanism for the reduction in NK cell number. Taken together, these three changes in leukocyte number induced by the protocol represent a remodeling of the immune systems of CMVþ subjects to look more like those of CMV subjects and successfully aging CMVþ centenarians.Physicians who monitored the health of the current study subjects through 1 year on the product reported no adverse events that were likely related to the protocol. However, 2 subjects who recently escalated their daily dose reported feeling ‘‘anxious’’ on 100 mg/day but not when they switched back to 50 mg/day. A placebo-controlled study will be needed to determine if this potential adverse effect is real. No new cases of cancer or cardiovascular disease were reported during the overall 260 person-years of dosing with PattonProtocol-1 through June, 2010, and this is statistically significant ( p< 0.05, cancer; p< 0.02, CVD ) assuming baseline age-specific risks in our population were similar to those of the U.S. population.TA-65 activated telomerase in cultured human cells at concentrations seen in the


plasma of subjects on the protocol. Paradoxically, although &40% of subjects showed an increase in mean telomere length over time, on average across all subjects there was a nonsignificant decline in mean telomere length. However, we speculate this effect is explained by cell dynamics and the fact that telomerase preferentially lengths the shortest telomeres.62–64 Rescue and selective expansion of near-senescent cells with short telomeres could lead to a reduction in the population mean telomere length despite some lengthening of telomeres in all cells. Because detrimental effects of telomere loss are primarily driven by short, dysfunctional telomeres, and loss of tissue function and disease onset in proliferative tissues have been associated with telomere lengths <4 kbp,25,65 we believe that our observed reduction in telomeres <4 kbp in subjects on PattonProtocol-1 is a significant, positive response, and that TA-65 contributes to the apparent benefit of the dietary supplement. In support of this, studies with TA-65 given orally in old mice showed similar reductions in percent cells with short telomeres and positive functional effects on tissues (Blasco et al., submitted) and preliminary dose–response analyses showed an increase in salutary effects with TA-65 doses up to 20–30mg per day average compared to the initial 5- to 10mg per day

Walter D. Funk,*,1 C. Kathy Wang,†,2 Dawne N. Shelton,* Calvin B. Harley,*Garrett D. Pagon,† and Warren K. Hoeffler†,‡

*Geron Corporation, 230 Constitution Drive, Menlo Park, California 94025; ‡Xgene Corporation, 863C Mitten Road,Burlingame,

California 94010; and †Department of Dermatology, Stanford University School of Medicine, Stanford, California 94305

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The lifespan of human fibroblasts and otherprimary cell strains can be extended by expressionof the telomerase catalytic subunit (hTERT). Sincereplicative senescence is accompanied bysubstantial alterations in gene expression, weevaluated characteristics of in vitro-aged dermalfibroblast populations before and afterimmortalization with telomerase. The biologicalbehavior of these populations was assessed byincorporation into reconstituted human skin.Reminiscent of skin in the elderly, we observedincreased fragility and subepidermal blistering withincreased passage number of dermal fibroblasts,but the expression of telomerase in late passagepopulations restored the normal nonblisteringphenotype. DNA microarray analysis showed thatsenescent fibroblasts express reduced levels ofcollagen I and III, as well as increased levels of aseries of markers associated with the destruction ofdermal matrix and inflammatory processes, andthat the expression of telomerase results in mRNAexpression patterns that are substantially similar toearly passage cells. Thus, telomerase activity notonly confers replicative immortality to skinfibroblasts, but can also prevent or reverse the lossof biological function seen in senescent cellpopulations.© 2000 Academic Press

Key Words: aging; telomerase; reconstituted skin;senescent fibroblasts; DNA microarray.

INTRODUCTION

Limited replicative potential is a definingcharacteristic of most normal human cells strains[1]. Within a population of cultured diploid cells,the proportion of actively dividing cells decreaseswith serial passaging while increasing numbers ofcells enter a state of terminal arrest, termedreplicative senescence. Similar

1 To whom reprint requestsshould be addressed. E-mail:

[email protected] Present address: Aviron, 297 North Bernardo

Avenue, Mountain View, CA 94043.

states of senescence arrest can be achieved by awide range of insults and effectors, includingactivated oncogenes [2–4], oxidative stressors [5,

6], chemical treatment [7, 8], and cdk inhibitors[9]; thus, the term cellular senescence moreaccurately defines a common terminal arrestphenotype triggered by numerous independentagents. Cellular senescence is also characterizedby an enlarged cell morphology, the activation ofa lysosomal b-galactosidase activity (senescence-associated (SA) b-galactosidase) [10], and alteredgene expression patterns [11–13] that maycontribute to pathologies associated with agingtissues and organs [14, 15].

The telomere hypothesis of replicativesenescence predicts that shortened telomericsequences trigger the senescence response inserially passaged normal cells [16, 17]. Inmammalian cells, telomeric sequences shortenwith each replication event and eventually,critically shortened telomeres trigger asenescence response. Telomere lengths aremaintained in cells that express telomerase [18], apolymerase activity that in human cellsminimally requires an RNA component, hTR[19], that most somatic cells express at lowlevels, and a catalytic protein component, hTERT[20, 21], whose expression correlates tightly withtelomerase activity. Germ cells, select stem cells,and the vast majority of tumor cells expresstelomerase, while most normal human cells donot. However, telomerase activity can bereconstituted in nonexpressing cells by forcedexpression of an hTERT transgene, resulting inlengthened telomeres and replicative immortality[22, 23]. As opposed to tumor lines, cellsimmortalized by hTERT retain normal growthcontrol and checkpoint mechanisms and a stablekaryotype [24, 25].0014-4827/00 $35.00 270Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

In this study, we sought to determine thecontribution of one senescent cell type, dermalfibroblasts, to the morphology and phenotype ofskin, and to determine the effects of lifespanextension by reexpression of telomerase. Using adermal reconstitution system, we compared thebehavior of telomerase-expressing, lifespan-extended cells to early passage and senescingcultures in functional assays and have evaluatedexpression patterns maintained by thesepopulations in vitro.


METHODS

Cell culture and retroviral transduction. BJ dermalfibroblasts (a gift from J. Smith, Baylor College of Medicine)were cultured in EMEM (GIBCO-BRL) with 10% fetalbovine serum (FBS) in humidified incubators at 37°C/10%CO2. Population doublings (PD) were determined by cellcounts upon passage. Replicative senescence in BJ cells isapparent near PD 90 (,5% S phase, doubling times greaterthan 3 weeks). Human foreskin keratinocytes were preparedfrom epidermis physically separated from dispase-treateddermis. The epidermis was minced with surgical forceps andthe tissue debris removed by passing the suspension through ametal mesh. Detached keratinocytes were pelleted,resuspended in 1:1 SFM (GIBCO-BRL) and KMK (Sigma)media, and plated on collagencoated dishes. Further culturesof primary keratinocytes were maintained in Keratinocyte-SFM medium (GIBCO-BRL) for a maximum of 5 PD inhumidified incubators at 37°C/10% CO2. For thereconstitution experiments, hTERT-expressing pBABEretrovirus and pBABE control retrovirus (a gift from W.Wright, University of Texas Southwestern Medical Center atDallas) were used to transduce BJ fibroblast cultures at PD89, which were then placed in selective medium containing0.3 mg/ml of puromycin. The resulting culture originatedfrom the expansion of several hundred transformed cells andwas passaged for an additional 20 population doublings aftertransduction.

Telomerase activity and telomere length analysis.Telomerase repeat amplification protocol (TRAP) assays wereperformed as described [26]. Mean telomere restrictionfragment (TRF) lengths were determined following Southernblot analysis of digested genomic DNA as described [22].

Dermal reconstitutions. All animal experiments wereconducted with the approval of the Animal Care Committee(APLAC) of Stanford University. Two-piece silicone culturechambers (CRD culture chambers, Renner, Germany) weresurgically implanted onto the backs of severe combinedimmunodeficient (SCID) mice to provide an aseptic woundbed resting on the muscle fascia. Dermal fibroblasts andkeratinocytes were harvested from culture by trypsinization,neutralized with PBS/10% FBS, and resuspended in serumfree medium (SFM, GIBCO). Human skin reconstitutionswere initiated by adding a mixed slurry of 6 3 106

keratinocytes and 6–8 3 106 dermal BJ fibroblasts to implantedchambers. After 1 week, the upper chambers were removed toallow for aeration of the skin surface. After 2 weeks, aconstant sheering force was applied to the area of thereconstitution using a rubberized mallet, as used bydermatologists to assess blistering potential. Immediatelyfollowing this treatment, the animals were sacrificed andreconstituted skin was harvested by surgical excision.

Microscopy and immunostaining. For hematoxylin/eosinstaining reconstituted skin samples were first fixed in 10%formaldehyde, embedded in paraffin, and then sectioned to 5-mm thickness cut normal to the cutaneous surface on aReichert-Jung 2040 microtome followed by staining withhematoxylin and eosin. For electron microscopy, sampleswere fixed in 2% paraformaldehyde, 2.5% glutaraldehyde,and 0.1 M cacodylate buffer, pH 7.4. Samples were treated

with 2% osmium tetroxide and 2% uranyl acetate, dehydrated,and embedded. For immunostaining, sections were fixed with220°C acetone for 10 min. Samples were rehydrated with fivesuccessive PBS washes. Blocking was conducted with mouseIgG diluted 1:400 (Jackson ImmunoResearch). Abiotin/avidin peroxidase conjugation system was used.Diluted primary antibody was incubated with the sample for 1h at room temperature, followed by three washes with PBS.Anti-laminin-5 b3 chain antibody K-140 was a gift from P.Marinkovich (Stanford University) and anti-collagen VIIantibody LH7:2 was a gift from I. Leigh (The Royal LondonHospital). An anti-mouse Ig–horseradish peroxidase conjugate(Amersham) was then incubated with the samples for 40 min.After three washes with PBS the samples were developedwith an insoluble peroxidase substrate (Sigma) for 20–30 min.The slides were lightly counterstained with hematoxylin,dehydrated, and mounted.

Microarray analysis. A custom DNA microarray wasproduced under contract with Incyte Microarray Services. Adetailed presentation of the composition and performance ofthis device can be viewed athttp://www.geron.com/pubsupplement/microarray.html.Approximately 1000 genes were selected and ESTscorresponding to these were identified by BLAST searches ofGenBank. The majority of the ESTs were I.M.A.G.E.consortium clones while others were identified from otherlibraries, or were available from existing plasmids. PCRamplification of the clones was performed with 59-aminemodified (Glen Research) oligonucleotidescomplementary to flanking sequences of the vector.Amplification products were analyzed by agarose gelelectrophoresis and then sent to the contractor for microarrayproduction. Poly(A1) mRNA was prepared from subconfluentcultures grown using OligoTex cartridges (Qiagen). The RNAwas quantified by A 260 measurements and assessed by agarosegel electrophoresis. Conversion of mRNA into either Cy5- orCy3-labeled cDNA probes and competitive hybridizations ofprobes were performed substantially as described [27] andbound signal was quantified by fluorescence measurement.Signals that scored with a signal to background level ,2.5 inboth channels were not considered. The total Cy5 signal wasnormalized to the total Cy3 signal and differential expressionratios were then calculated. Each experimental pairing ofmRNA was performed in duplicate and the results present theaverage of the two measurements. The identities of all genesindicated in this report were confirmed by DNA sequenceanalysis of the corresponding cDNAs.

RESULTS

hTERT Expression Restores Replicative Capacityto

Late Passage Fibroblast Populations

We first documented the ability of hTERTexpression to rescue the replicative potential ofour late passage BJ fibroblast population at PD89. Untreated or pBABE control-transduced

26 FUNK ET AL.

cultures did not express telomerase activity (Fig.1a), and had mean TRF lengths that were muchshorter than those of early passage BJ cells (Fig.1b). Senescence of these cultures was apparent atPD 93, at which point S-phase cells were reducedto less than 5%, and the population failed todouble within a 3-week period. Culturestransduced at PD 89 with hTERT-expressingretrovirus expressed detectable telomerasecatalytic activity. The lengthening of the meanTRF of the telomerase-expressing DS-1 line wasobserved to increase once the culture hadsurpassed the normal senescence point,suggesting that only those cells that had restoredtelomeres were capable of extended lifespan. DS-1 cells showed no signs of reduced growth at PD110 (Fig. 1c), and similar hTERT-expressing BJcultures have been grown to at least PD 280 [24].

FIG. 1. Telomerase activity and telomere length analysis. (a) TRAP assays were performed on cell extracts prepared from mass culture late passage BJfibroblasts, and late passage fibroblasts transduced at PD89 with pBABE control or pBABE-hTERT retroviral vectors. IC, internal control for PCR portionof TRAP assay. (b) Southern blot analysis for terminal restriction fragment (TRF) lengths was performed for mass culture BJ fibroblasts at early and latepassage, and for control- or hTERT-transduced cultures. (c) Growth curves for late passage BJ (‚), pBABE control (following retroviral infection. PD, population doubling.

Sheer-Sensitivity of Skin Reconstitutions Performed with Senescent Fibroblasts Is Not Seen in Reconstitutions with TERT-ExpressingFibroblasts

Tissue-related characteristics of BJ fibroblastswere assessed by incorporating these cells intohuman skin reconstitutions. A variety of modelsare available for this purpose. We chose avariation of a method that gives rise to epidermaland dermal layers, where striation ofimmunohistological markers in the reconstitutedskin model are identical to normal skin [28]. Themethod utilizes a silicone chamber [29, 30]implanted directly on mouse muscle fascia for theincubation of human keratinocytes andfibroblasts. Over the course of 2 weeks, thefibroblast and keratinocyte populations segregateinto dermal and epidermal layers and form adermal composite that closely matches humandermal architecture [28]. We compared directlythe morphology of reconstituted human skincreated using early (PD ;20), middle (PD ;60),and late passage (PD ;85) BJ dermal fibroblasts,and the telomerase positive DS-1 strain (PD ;110). Light microscopy of hematoxylin/eosin(H/E)-stained paraffin-embedded thin sections ofthe early passage reconstitutions revealed anormal, tightly joined epidermis and dermis (Fig.2a). In contrast, intermittent splitting (blistering)near the dermal–epidermal (D/E) junction wasobserved in the middle passage reconstitution(Fig. 2b), while extensive splitting was observedin reconstitutions performed with the late passagefibroblasts (Fig. 2c). The proportion of senescentcells, as judged by SA b-galactosidase staining,increases with serial passage [10] and it is likelythat the observed defects evident in middle andlate passage reconstitutions reflect thisaccumulation. Reconstitutions performed with thetelomerase-expressing DS-1 strain, which did notstain significantly for SA b-galactosidase activity(data not shown), demonstrated skin integritynear the D/E junction that was similar to thatobserved with early passage cells (Fig. 2d). Boththe early and DS-1 reconstitutions were amenableto a higher resolution analysis

FIG. 2. Splitting (blistering) of reconstituted human skin increases with serial passaging of fibroblasts and is prevented bytelomerase expression. Results are representative of repeat experiments involving a total of at least six animals for early,


middle, and late passage reconstitutions, and three animals for the DS-1 reconstitutions. Hematoxylin/eosin-stained thin sectionsare shown for: (a) early passage BJ skin fibroblasts (PD ;20); (b) middle passage BJ skin fibroblasts (PD ;60); (c) late passagefibroblasts (PD ;85); (d) DS-1 cells (PD 110); electron micrographs (61,3003) of human skin reconstitutions performed with (e)early passage (PD 20) BJ fibroblasts or (f) DS-1 fibroblasts (PD 110) expressing a telomerase transgene. Bars on the right

indicate extent of epidermal keratinocytes (white) and dermal fibroblasts (black). Note that hemidesmosomes linked intointermediate filaments within the epidermis are positioned directly across from wispy shaded regions that contain collagenfilaments (arrows).

28 FUNK ET AL.

by electron microscopy (EM) of the dermal–epidermal precluded the possibility of further analysis.The high junction, since these junctions remained intact, resolution of the EMs allow assessment ofultrastrucwhereas splitting of the junctions in the later samples tural elements present within thedermal–epidermaljunction between single cells. Both sections from reconstitutions performed with early passage BJfibroblasts or DS-1 fibroblasts revealed very similar ultrastructures including hemidesmosomes withattached intermediate filaments within the epidermal cells, with connections to collagen filaments seenon the dermal fibroblast side of the junction (Figs. 2e and 2f).

Sheer-Sensitive Splitting in Senescent Fibroblast Reconstitutions Occurs in the Dermal Layer

To define more precisely the splitting observed in reconstitutions performed with later passagefibroblasts, immunohistological staining was conducted for proteins known to reside at discrete layerswithin normal skin. Laminin-5 is the main component of anchoring filaments that are a part of thehemidesmosomes, ultrastructures responsible for connecting dermal and epidermal layers.Immunohistological staining with a laminin-5 antibody showed that the split occurs below the level ofthe D/E junction, since all of the laminin-5 remained attached to the epidermal side of the split (Fig.3a). Likewise, immunohistological staining using an antibody specific for collagen VII also illustratedthat the split is below the anchoring fibrils, structures localized entirely in the dermis, since all of thecollagen VII remained on the epidermal side of the split (Fig. 3b). The deficient componentresponsible for the compromised integrity of the reconstituted skin must reside below the D/E junctionitself. This conclusion is further supported by the observation that occasionally a few fibroblasts arefound to remain adhered to the D/E junction, indicating that the split is due to a deficiency below thejunction.

Senescence-Associated Gene Expression Patterns Are Substantially Prevented by Telomerase Expression

To assess the alterations in gene expression at senescence, we developed a custom DNA microarray[13] and used it to contrast early, late, and telomeraseexpressing passages of BJ fibroblasts. Comparedto early passage cells, BJ fibroblasts at senescence overexpress markers associated with a variety ofprocesses (Fig. 4a). The cdk inhibitor p21, and growth arrestspecific (gas1) mRNA are likelyparticipants, or markers, of cell cycle arrest, while the expression of chemokines MCP-1, Gro-a,cytokines Il-1b, Il-15, and the adhesion molecule ICAM-1 are characteristic of an inflammatoryresponse. Matrix-degrading activities are well-documented characteristics of senescent cells [11] asdemonstrated here by the expression of tPA, stromelysins-1,2, and cathepsin O, while stress-associated genes, such as GADD153 and MnSOD, are also upregulated. Many of these proteolytic andchemotactic activities are also associated with wound repair andserum responsiveness of normal dermal fibroblasts [13, 31] and suggest that senescent cells are lockedinto an inappropriate, proinflammatory state, perhaps preventing the later anabolic phase of normalwound healing [13].In DS-1 cells, assessed 17 PD past the normal point of replicative exhaustion, the majority of thesemarkers are expressed at levels comparable to early passage cells. Specifically, mRNA levels ofproinflammatory molecules, matrix proteases, and stress-associated genes returned to levelscomparable to early passage cells, and a similar response was seen for most, but not all, of the othermarkers associated with senescence (Fig. 4a). These results demonstrate that many of these changes ingene expression associated with replicative senescence can be prevented by the expression oftelomerase.

We also examined the expression of genes whose expression are repressed at senescence, inparticular genes involved in deposition and maintenance of the dermal extracellular matrix (ECM).The primary ECM material is collagen I, making up about 77% of the dry weight of skin and, together


with collagen III and elastin, makes up the majority of the ECM. The expression of collagen 1a1 andcollagen IIIa1 is low in senescent cells [32, 33] but in DS-1 cells these levels are restored to thoseseen with early passage BJ fibroblasts (Fig. 4b). The expression of elastin is also low in senescentcells, while in DS-1 cells expression does not appear to be substantially changed as a result oftelomerase expression.

FIG. 3. The level of the split is below laminin-5 and collagen VII. Immunostainingwas performed on dermal reconstitutions using middle passage fibroblasts (PD ;60)(a) Laminin-5, a component of the hemidesmosomes, is localized between dermaland epidermal layers, and stains along the upper blister surface indicating asubepidermal split, arrow. (b) Collagen VII, a component of the anchoring filamentsthat are in the upper dermal surface, also stains along the upper surface of theblister indicating that the split occurs below the dermal/epidermal junction, arrow.

FIG. 4. DNA microarray analysis of mRNA levels in early passage and late passage BJ fibroblasts and in telomerase-expressing DS-1 fibroblasts. (a) Genes overexpressed in late passage fibroblasts. Black bar: Fold-differential expression ofmRNA in senescent BJ cells (PD 92) relative to early passage BJ cells (PD 30). Gray bar: Expression of same genes intelomerase-expressing DS-1 cells (PD 110) relative to early passage BJ cells. (b) Genes overexpressed in early passagefibroblasts. Black bar: Fold-differential expression of mRNA in early passage BJ cells relative to senescent BJ cells. Gray bar:Expression of same genes in DS-1 cells relative to senescent passage BJ cells.

DISCUSSION

To date, cellular senescence has been studied primarily in the context of culturedhuman cell lines, while the biological consequences of this condition remain largelyunexplored. Data supporting the increasing frequency of senescent cells in humanskin with chronological aging have been reported [10] and our study provides directevidence that the senescent phenotype results in structural deficits in reconstitutedhuman skin and that telomerase expression is able to restore biological function tolate passage cell strains.

Cellular Senescence, Skin Aging, and StructuralDefects

The skin reconstitution model used in this study provides an accurate facsimile ofhuman dermis and can be particularly useful in contrasting the behavior of donorcells. Our examination of the effects of senescence in fibroblast populations showedsignificant weakening of the structural integrity of the dermal matrix, a finding that isconsistent with thinning of the matrix that occurs with chronological aging [15]. Thereconstitution system may model more accurately wound healing processes, in thatcell populations are required to repopulate and remodel a full-thickness dermaldeficit. In mice lacking telomerase activity due to a knockout of the mTR locus,

telomeres shorten and dermal pathologies are observed that include ulcerativelesions at sites of high mechanical stress and reduced wound repair [34],observations that are consistent with the mechanical fragility observed in latepassage reconstitutions. The skin organ contains many additional cell types, such asLangerhans cells, melanocytes, mast cells, and endothelial cells, and these are notprovided in our reconstitutions. Aspects likely to be specific to keratinocytes andfibroblasts, respectively, include fragility of the skin along the D/E junction resultingin blistering, a decline in the thickness of the dermis, disorganization of collagenbundles and elastin fibrils in the dermis, and a decrease in the turnover rate forkeratinocytes [35].

Expression of hTERT results in the rescue of replication-competent cells even whentransducing very late passage populations. Retroviral transduction is selective foractively dividing cells and thus it is possible that the DS-1 population derived fromthe few remaining actively dividing cells in the original senescing population but notfrom the transduction of arrested cells. The extent to which telomerase expression iscapable of restoring the molecular phenotype of late passage cells to that of earlypassage cells may reflect two processes. First, a portion of what is described as thesenescent phenotype may include long-term cumulative defects, such as oxidativedamage to mitochondria, and these changes may not be affected by telomeraseexpression. Whatever the nature of any accumulated damage in long-term culture,such deficits clearly do not accumulate to a degree necessary to affect proliferativecapacity, since telomerase-immortalized cultures have been maintained for hundredsof population doublings. Second, long-term cultivation of cells in vitro may result inthe selection of long-lived subpopulations of cells whose expression patterns differintrinsically from that of the early passage culture because of phenotypic drift. Forexample, the expression of elastin in DS-1 populations is not restored to levels seenin early passage cells, while levels of IGFBP5 remain elevated. The ability of IGF-BPsto inhibit the action of IGF, a known inducer of elastin expression [36], may accountfor this finding. Regardless, the majority of changes in gene expression observed insenescent populations are not observed in telomeraseimmortalized cells, suggestingthat most gene dysregulation associated with replicative senescence results directly,or indirectly, from telomere attrition.

Gene Expression Changes at Senescence

The pronounced proinflammatory response of dermal fibroblasts at senescence inmany ways resembles an activated state associated with the early phases of woundhealing. The potent mix of cytokines and chemokines elicited by senescent cells canmobilize immune response cells, while proteolytic activities, such as plasminogenactivators and stromelysins, play a significant role in the breakdown of fibrin clotsand dermal matrix. Expression of the prohormone converting enzyme peptidyl-amidating monooxygenase (PAM) is also consistent with a wound-activatedphenotype, as expression of neuropeptides has been documented to participate indermal inflammatory processes [37]. As assessed by this broad survey of transcripts,the expression of these transcripts is suppressed in telomerase-expressingpopulations to levels consistent with those of early passage cells, suggesting stronglythat their elevated expression in late passage is correlated primarily with thetelomere-related senescence arrest, and not due to effects of long-term culture or

generalized damage.One of the outstanding issues regarding cellular senescence remains the extent to

which this process initiates or contributes to disease processes, particularly thoseassociated with human aging. Experimental evidence showing an increasedabundance of SA-b-galactosidase-positive cells in human dermis with donor age hasbeen reported [10]; however, the inflammatory transcript profile provided bysenescent fibroblasts might suggest that these cells would be eliminated from tissueby immune responses. Regardless, the senescent phenotype may still contribute topathologies, since many of the effects predicted by gene expression analyses wouldbe extracellular and could affect tissue integrity. The results from our dermalreconstitution studies suggest a deficit in wound healing in tissues with increasedabundance of senescent, or near senescent, cells. In support of this, an increasedoccurrence of senescent fibroblasts has been observed in chronic wound biopsies[33, 39], and age significantly effects the efficiency of wound healing in aeschemiculcer models [40].

The relationship of telomerase-extended cellular lifespans to cancer have yet to befully defined, but the expression of telomerase in human cells does not alter theirresponse to a variety of cell cycle arrest effectors, nor does it result in malignanttransformation or genotypic instability [24, 25]. Recent results with human vascularendothelial cells [41] and bovine adrenocortical cells [42] have shown that earlypassage cells expressing telomerase expression retain complex biological functionand the results shown here indicate that telomerase expression, even at very latestages of passage, prevents the majority of alterations in gene expression associatedwith senescence. The potential of utilizing telomerase activation to treat cellularagerelated conditions ex vivo and in vivo is under investigation.

We thank D. Choi for the TRF analysis and P. Whittier for assistance with themicroarray sample preparations.

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